The chemical compositions and anti-tumor activities of the petroleum ether fraction PE, from mushroom Pyropolyporus fomentarius, were studied. Upon gas chromatography–mass spectrometry GC–MS analysis, nine major constituents were identified in the fraction. In vitro, the PE showed cytotoxic activity against murine sarcoma S180 S180 cells in a dose- and time-dependent manner, and the cytotoxic effects were associated with apoptosis. The mitochondrial membrane potential loss and the intracellular ROS generation were greatly increased in the Pyropolyporus fomentarius PE treated group, suggesting cell apoptosis, induced by the PE in S180 cells, might be mitochondria dependent and ROS mediated. Consistent with in vitro findings, the in vivo study showed that the Pyropolyporus fomentarius PE was also effective in inhibiting the tumor growth induced by S180 cells and had lower immune organ toxicity. We found that the Pyropolyporus fomentarius PE has significant anti-tumor activity and great potential in screening anti-tumor drugs.